The principal objectives of the proposed investigation are: 1) to complete the purification of the substance(s) comprising the pheromonal system emanating from the female Asian elephant urine prior to ovulation, using the sequential high recovery fractionation procedures devised during NIH grant #HD 19219-02 (1984-87). The proven reliable bioassay involving the flehmen response by bulls will continue to be used to detect pheromones present in estrous urine fractions and to guide the fractionation procedures; 2) to determine the chemical and structural nature of the purified molecule(s). Currently the active fraction obtained after a procession of steps including successvie high performanc liquid chromatographic separations, while purified a thousand fold, contains multiple components. To provide sufficient material for final purification, the quantities of bioactive urine processed have been scaled up another 5 fold. The separation scheme has been improved by insertion of a G-25 Sephadex chromatography step prior to the initial high performance liquid chromatography fractionation. Dr. Terry Lee, Beckman Research Institute, using HPLC systems of higher resolution and capacity, will undertake late stage HPLC fractionation. Structural identification will be undertaken when sufficiently homogeneous bioactive material is available using fast atom bombardment mass spectrometry (FABMS), nuclear magnetic resonance spectrometry, compound derivization, and other chemical procedures (e.g. amino acid sequencing) as warranted. The collaboration with Dr. Terry Lee, Beckman Research Institute and Dr. G. D. Daves, Lehigh University, insures both access to state-of-the-art tools for structural analyses and to expertise in structural identification. Molecular and structural identification of the pheromone will make possible a more precise assessment of its role in reproductive physiology and chemical communication involving the vomeronasal organ, an organ associated with brain centers implicated in male sexuality. Post-identification problems include: measurement of pheromone concentrations relative to estrogen and/or progesterone levels and relative to individual female elephants, and the comparison of male serum luteinizing hormone and testosterone levels, measured by radioimmunoassay, with frequency of cow access (probing the possibility of a releaser or primer system).